1. Field of the Invention
The present invention relates generally to the fields of molecular biology and protein synthesis. More particularly, it concerns methods of increasing production of biologically functional nitric oxide synthase and other proteins that require specific post-translational alteration for activity.
2. Description of the Related Art
Nitric oxide synthase (NOS) catalyzes the formation of NO. and L-citrulline from L-arginine through a series of oxidations using molecular oxygen (Bredt and Snyder, 1990). There are at least three separate genes encoding the NOS family of proteins, including the constitutively expressed neuronal (nNOS) (Bredt et al.. 1991) and endothelial (ecNOS) (Lamas et al., 1992; Sessa et al., 1992) isoforms, and the inducible isoform (iNOS) (Xie et al., 1992; Lowenstein et al., 1992).
The three isoforms differ in primary sequence (having only 50-60% sequence identity), size, intracellular location, and regulation. Neuronal NOS (160 kDa) and iNOS (130 kDa) were purified from the cytosol (Masters, 1994; Bredt and Snyder, 1994; Schmidt et al., 1991; Hevel et al., 1991; Stuehr et al., 1991), whereas the ecNOS (135 kDa) was found to be membrane-bound (Pollock et al., 1991). The nNOS and ecNOS are constitutively expressed, but modulated by intracellular Ca.sup.2+ levels (Bredt and Snyder, 1990; Forstermann et al., 1991) unlike iNOS which is induced by bacterial endotoxin and is Ca.sup.2+ -independent (Cho et al., 1992). All three isoforms contain calmodulin and tetrahydrobiopterin (BH.sub.4), as well as molar ratios of heme, FMN, and FAD (Hevel et al., 1991; Stuehr et al., 1991; Pollock et al., 1991; Mayer et al., 1991; White and Marletta, 1992; Stuehr and Ikeda-Saito, 1992; McMillan et al., 1992; Klatt et al., 1992). These members of the NOS family are unusual mammalian enzymes in that they catalyze both NADPH-mediated reduction of flavins and heme within the same protein. A cytochrome P450 fatty acid hydroxylase in Bacillus megaterium containing both flavins, FAD and FMN, in the same polypeptide chain as the heme has been reported by Narhi and Fulco (1986). Nitric oxide synthases, however, represent the first examples of such complex enzymes from mammalian sources.
Nelson et al. (1993) reportedly describes a relationship between the P450 gene superfamily and the nitric oxide synthase genes as a likely example of convergent evolution. While the nitric oxide synthases contain a carboxyterminal domain with sequence similarity to NADPH-P450 oxidoreductase (Bredt et al., 1991), and also possess heme and other biochemical features indicative of a P450-like function (White & Marletta, 1992), the nitric oxide synthases lack the P450 ten amino acid signature sequence present in 202 of 205 sequences compared in Nelson et al., 1993 and do not exhibit the characteristic I-helix present in all known cytochromes P450 (McMillen et al., 1992). Based on sequence alignments, the percent identity between mouse nitric oxide synthase and 57 cytochrome P450s was an average of 11.6%, about what would be expected for random amino acid sequences (Nelson et al., 1993).
Mechanistic and structure/function studies of nNOS have been very difficult to conduct due to the minute amounts of protein that can be purified from cerebellar tissue. Bredt and Snyder (1990) reported a yield of 9 .mu.g of pure protein from 18 rat brains. Bredt, et al. (1991) subsequently cloned and expressed nNOS in human kidney 293 cells, providing a 10-fold enrichment of nNOS in cultured cells over rat brain. The expense and time involved in mammalian cell culture, however, are prohibitive for generating large amounts of enzyme. Other laboratories have expressed nNOS using baculovirus overexpression systems. Charles, et al. (1993) report successful expression, but the majority of this recombinant nNOS is insoluble and inactive; the recombinant enzyme has a specific activity that is 100-fold lower than that of native nNOS isolated from rat cerebellar tissue. Richards and Marletta (1994) improved the yield of active enzyme from the baculovirus system by adding hemin to the media, but still isolated only about 1 mg pure protein from seven to ten 75 cm.sup.2 monolayer cultures, only about half of which contains heme. More recently, however, Riveros-Moreno et al. (1995) have been more successful with the baculovirus expression system, producing 30 mg of highly active enzyme per 17-225 cm.sup.2 flask with one liter of media.
Nitric oxide is a short-lived molecule with a large number of roles, most of which are involved in signalling in the nervous and cardiovascular systems. In these cases, NO. acts by activating a soluble guanylate cyclase in the target cell, which leads to an intracellular accumulation of cyclic 3',5' guanosine monophosphate. This messenger molecule then activates a cascade of intracellular enzymes to bring about the biological effect, which ranges from relaxing isolated blood vessels to neurotransmission to neurodegeneration associated with decreased blood flow in AIDS dementia and Parkinson's disease (Ogden and Moore, 1995). In addition, NO. may act as a powerful reactive free radical that may contribute to the cytostatic and neurodegenerative effects of NO..
It is therefore advantageous, in certain circumstances, to inhibit the action of NOS as a means of reducing NO. production. Potential advantages of such inhibition include reducing the blood pressure loss that is associated with endotoxic shock, which has been shown to be associated with a large release of NO. arising from the cytokine-inducible NOS of macrophages. These large amounts of NO. have been shown to cause hypotension from uncontrolled vasorelaxation of vascular smooth muscle cells. Other investigators have shown that the selective inhibition of iNOS may be beneficial in treating some forms of inflammatory diseases.
Because numerous potential clinical applications for selective inhibition of both nNOS and iNOS isoforms have been identified, it is therefore advantageous to screen for inhibitors of NOS that will allow the development of new classes of compounds to block the deleterious effects of NO. in selected cells. While several techniques are known for screening for NOS inhibitors, a reliable and relatively inexpensive source of single isoform NOS has heretofore been unavailable.
Problems with the prior art methods of producing NOS include: i) insufficient amounts of protein produced, ii) expense and time involved in mammalian cell culture, iii) insolubility of expressed protein ("inclusion bodies"), iv) inactivity, v) low specific activity, and vi) insufficient incorporation of the many cofactors that are required for enzymatic activity. Because of all of the above problems, known procedures are not completely satisfactory, and persons skilled in the art have continued to search for improvements.